Antifungal Activities of Essential Oils from Thymus quinquecostatus and T. magnus

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

The antifungal activities of essential oils from Thymus quinquecostatus and T. magnus, which are species native to Korea, were evaluated against seven common pathogenic fungi. Additionally, the effects of the oils together with ketoconazole were tested by the checkerboard titer test. Both of the two Thymus oils showed significant inhibition of the tested fungi, with minimal inhibitor concentrations (MICs) and minimal fungicidal concentrations (MFCs) in the range of 0.04 - 0.39 mg/mL and 0.19 - 0.78 mg/mL, respectively. The two Thymus oils, and thymol as well, exhibited synergism with ketoconazole against Trichophyton rubrum, which showed the highest susceptibility to these oils.

The development of natural antifungal agents is appealing because of the problematic toxicity, rapid development of resistance, and drug-drug interactions associated with the therapeutics currently used to treat mycoses. Many volatile oils are potent sources of natural antifungal chemicals even though they have relatively mild activities compared to the present synthetic therapeutics [1], [2]. Thymus species are well known sources of antimicrobial essential oils and vary tremendously in composition depending on the plant source [3], [4], [5], [6]. T. quinquecostatus Celakov and T. magnus Nakai (Labiatae) contain high percentages of thymol. They are native to Korea and they have been used as diaphoretics and carminative in traditional medicine [7], [8]. In this study the antifungal activities of the essential oils from T. quinquecostatus and T. magnus as well as their main constituents were evaluated against eight common pathogenic fungi. The compositions of the essential oils were analyzed and compared by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS).

The oil compositions were generally consistent with previously reported compositions [8] except for thymol levels which were higher in our study (Table [1]). In both of the oils, p-cymene, γ-terpinene and camphor (only in T. magnus) were the most prominent components after thymol.

In the broth dilution test, the two Thymus oils showed significant inhibition against the test fungi, with MICs and MFCs in the range of 0.04 - 0.78 mg/mL (Table [2]). Generally, there were no remarkable differences between the two oils with respect to their antifungal activity; however, the oil from T. quinquecostatus showed slightly higher activity against T. rubrum with a two-fold lower MIC (0.04 mg/mL) compared to the MIC (0.09 mg/mL) of the oil from T. magnus.

The activities of thymol and carvacrol were similar to that of the total oils of the two Thymus species against most of the fungi tested, while the other main components, p-cymene and γ-terpinene, had relatively mild activities, with MICs from 0.19 to > 6.25 mg/mL and MFCs from 0.39 to > 6.25 mg/mL. Camphor showed a higher activity than p-cymene or γ-terpinene, albeit much lower than that of thymol. These results indicate that the minor components also contribute to the activities of the total oils. T. rubrum showed the highest susceptibility, with MICs from 0.04 to 0.19 mg/mL, to all of the tested oil samples. Additionally, when calculated by the checkerboard titer test, the two Thymus oils, as well as thymol, exhibited significant synergism [2] with ketoconazole against T. rubrum, resulting in the same fractional inhibitory concentration indices (FICI) of between 0.35 and 0.37. Furthermore, the results of the checkerboard titer test depicted in the isobologram in Fig. [1] confirm the synergism between T. magnus oil and ketoconazole, as the combination of both compounds produced a curve that had shifted to the left of the line (dotted) of the added effects of the two compounds employed separately. Therefore, these oils might be useful in antifungal therapy in combination with ketoconazole, especially against T. rubrum. However, further research is required to assess their value in practical applications.

Materials and Methods

The essential oils were obtained by steam distillation for five hours in a simultaneous steam distillation-extraction apparatus from flowers and leaves (1 : 1) of cultivated T. quinquecostatus and T. magnus harvested in July from the herbal garden of Duksung Women’s University. The plants were identified by Prof. C. M. Kim (College of Pharmacy, Kangwon National University, Chunchun, Korea) and voucher specimens were deposited at the herbarium of Duksung Women’s University (No. LABT1 and LABT2). The essential oil fractions were analyzed by a Hewlett-Packard 6890 GC and a Hewlett-Packard 5973 MSD apparatus (Agilent 5973 network mass selective detector, 280 °C) with a fused silica capillary column with two different stationary phases (HP-1 and HP-Innowax, 30 m × 250 μm × 0.25 μm), respectively. The injector was adjusted to 250 °C and the oven temperature was programmed as follows: Initial temperature: 50 °C for 5 min, 2 °C/min up to 180 °C, 5 min at 180 °C, and then 20 °C/min up to 220 °C.

The test fungi were subdivided from the Korean Culture Center of Microorganisms (KCCM). Thymol (98 %), p-cymene (99 %), g-terpinene (97 %), and ketoconazole (98.8 %) were purchased from Sigma Chemical Co. The broth dilution test to determine the MICs and MFCs and the checkerboard titer tests were performed as previously reported [9]. Each organism was also cultured with a blank solution containing ethanol and Tween 80 at concentrations equivalent to those in the test solutions to certify that these vehicles did not affect fungal growth. MIC and MFC values were tested in duplicate and were retested if the values differed. In the checkerboard titer tests fractional inhibitory concentrations (FICs) were calculated as the MIC of the combination of ketoconazole and the essential oil of thymus, or thymol, divided by the MIC of the oil or ketoconazole alone. The FIC index was calculated by adding both FICs and was interpreted as a synergistic effect when it was ≤ 0.5, as additive or indifferent when it was > 0.5 to 2.0 and as antagonistic when it was > 2.0. An isobologram was constructed on the basis of the checkerboard experiment to depict the synergism of the oils with ketoconazole against the fungi.

Acknowledgements

This study was supported by grant no. R04-2002-000-00 058-0 from the Korea Science and Engineering Foundation.

References

• 1 Hammer K, Carson C F, Riley T V. In vitro activities of ketoconazole, econazole, miconazole, and Melaleuca alternifolia (tea tree) oil against Malassezia species.  Antimicrob Agents Ch. 2000;  44 467-9
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• 3 Zarzuelo A, Crespo E T. The medicinal and non-medicinal uses of thyme. In: Thyme-the genus Thymus . Stahl-Biskup E, Saez F. editors London, New York; Taylor & Francis 2003: pp 263-92
  Search in Google Scholar
• 4 Rustaiyan A, Masoudi S, Monfared A, Kamalinejad M, Lajevardi T, Sedaghat S. et al . Volatile constituents of three Thymus species grown wild in Iran.  Planta Medica. 2000;  66 197-8
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• 5 Mikus J, Harkenthal M, Steverding S, Reichling J. In vitro effect of essential oils and isolated mono- and sesquiterpenes on Leishmania major and Trypanosoma brucei .  Planta Medica. 2000;  66 366-8
  Thieme ConnectPubMedSearch in Google Scholar
• 6 Faleiro M L, Miguel M G, Ladeiro F, Venâncio F, Tavares R, Brito J C. et al . Antimicrobial activity of essential oils isolated from Portuguese endemic species of Thymus .  Lett Appl Microb. 2003;  36 35-40
  PubMedSearch in Google Scholar
• 7 Shin H, Choi H K. A taxonomic study on Thymus in Korea: Numerical analyses of morphological characters.  Kor J Plan Tax. 1997;  27 117-35
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• 8 Kim Y H, Lee J C, Choi Y H. Essential oils of Thymus quinquecostatus Celakov. and Thymus magnus Nakai.  Korean J Medicinal Crop Sci. 1994;  2 234-40
  PubMedSearch in Google Scholar
• 9 Shin S, Kang C A. Antifungal activity of the essential oil of Agastache rugosa Kuntze and its synergism with ketoconazole.  Lett Appl Microb. 2003;  36 111-5
  PubMedSearch in Google Scholar

Prof. Seungwon Shin, Ph. D.

College of Pharmacy

Duksung Women’s University

Ssangmoondong 419

Dobongku

Seoul 132-714

Korea

Phone: +82-2-901-8384

Fax: +82-2-901-8386

Email: swshin@duksung.ac.kr

References

• 1 Hammer K, Carson C F, Riley T V. In vitro activities of ketoconazole, econazole, miconazole, and Melaleuca alternifolia (tea tree) oil against Malassezia species.  Antimicrob Agents Ch. 2000;  44 467-9
  PubMedSearch in Google Scholar
• 2 Shin S. Anti-Aspergillus activities of plant essential oils and their combination effects with ketoconazole or amphotericin B.  Arch Pharm Res. 2003;  26 389-93
  CrossrefPubMedSearch in Google Scholar
• 3 Zarzuelo A, Crespo E T. The medicinal and non-medicinal uses of thyme. In: Thyme-the genus Thymus . Stahl-Biskup E, Saez F. editors London, New York; Taylor & Francis 2003: pp 263-92
  Search in Google Scholar
• 4 Rustaiyan A, Masoudi S, Monfared A, Kamalinejad M, Lajevardi T, Sedaghat S. et al . Volatile constituents of three Thymus species grown wild in Iran.  Planta Medica. 2000;  66 197-8
  PubMedSearch in Google Scholar
• 5 Mikus J, Harkenthal M, Steverding S, Reichling J. In vitro effect of essential oils and isolated mono- and sesquiterpenes on Leishmania major and Trypanosoma brucei .  Planta Medica. 2000;  66 366-8
  Thieme ConnectPubMedSearch in Google Scholar
• 6 Faleiro M L, Miguel M G, Ladeiro F, Venâncio F, Tavares R, Brito J C. et al . Antimicrobial activity of essential oils isolated from Portuguese endemic species of Thymus .  Lett Appl Microb. 2003;  36 35-40
  PubMedSearch in Google Scholar
• 7 Shin H, Choi H K. A taxonomic study on Thymus in Korea: Numerical analyses of morphological characters.  Kor J Plan Tax. 1997;  27 117-35
  PubMedSearch in Google Scholar
• 8 Kim Y H, Lee J C, Choi Y H. Essential oils of Thymus quinquecostatus Celakov. and Thymus magnus Nakai.  Korean J Medicinal Crop Sci. 1994;  2 234-40
  PubMedSearch in Google Scholar
• 9 Shin S, Kang C A. Antifungal activity of the essential oil of Agastache rugosa Kuntze and its synergism with ketoconazole.  Lett Appl Microb. 2003;  36 111-5
  PubMedSearch in Google Scholar

Prof. Seungwon Shin, Ph. D.

College of Pharmacy

Duksung Women’s University

Ssangmoondong 419

Dobongku

Seoul 132-714

Korea

Phone: +82-2-901-8384

Fax: +82-2-901-8386

Email: swshin@duksung.ac.kr